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Resistance to key first-line drugs is a major hurdle to achieve the global end tuberculosis (TB) targets. A prodrug, pyrazinamide (PZA) is the only drug, effective in latent TB, recommended in drug resistance and susceptible Mycobacterium tuberculosis (MTB) isolates. The prodrug conversion into active form, pyrazinoic acid (POA), required the activity of pncA gene encoded pyrazinamidase (PZase). Although pncA mutations have been commonly associated with PZA resistance but a small number of resistance cases have been associated with mutationss in RpsA protein. Here in this study a total of 69 PZA resistance isolates have been sequenced for pncA mutations. However, samples that were found PZA resistant but pncA wild type ( pncA WT ), have been sequenced for rpsA and panD genes mutation. We repeated a drug susceptibility testing according to the WHO guidelines on 18 pncA WT MTB isolates. The rpsA and panD genes were sequenced. Out of total 69 PZA resistant isolates, 51 harbored 36 mutations in pncA gene (GeneBank Accession No. MH46111) while, fifteen different mutations including seven novel, were detected in the fourth S1 domain of RpsA known as C-terminal (MtRpsA CTD ) end. We did not detect any mutations in panD gene. Among the rpsA mutations, we investigated the molecular mechanism of resistance behind mutations, D342N, D343N, A344P, and I351F, present in the MtRpsA CTD through molecular dynamic simulations (MD). WT showed a good drug binding affinity as compared to mutants (MTs), D342N, D343N, A344P, and I351F. Binding pocket volume, stability, and fluctuations have been altered whereas the total energy, protein folding, and geometric shape analysis further explored a significant variation between WT and MTs. In conclusion, mutations in MtRpsA CTD might be involved to alter the RpsA activity, resulting in drug resistance. Such molecular mechanism behind resistance may provide a better insight into the resistance mechanism to achieve the global TB control targets.

The cotton whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a serious pest of several summer crops in hot and dry climates. Its field populations (Asia II-1 biotype) were assessed for their susceptibility to diverse pesticides by using leaf-dip bioassay. There was no or a very low resistance to amitraz, hexythiazox, and pyridaben during 1992–2015. B. tabaci also exhibited no resistance to endosulfan during 1992–1997 and a very low resistance during 1998–2010, which then rose to a low level during 2011–2015. Chlorfenapyr resistance was very low during 1997–2008 and it reached to a high level during 2009–2011 and to a very high level in 2013 and 2015. Among avermectins, abamectin showed a very low resistance up to 2013 but a high resistance in 2015. Emamectin benzoate also demonstrated a very low resistance up to 2010, but a moderate-to-high resistance during 2011–2015. It may be concluded that the diverse chemistries, having novel modes of action and showing no, very low or low levels of resistance, can be substituted in rotation in the wake of resistance development to conventional insecticides.

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) belongs to the single-stranded positive-sense RNA family. The virus contains a large genome that encodes four structural proteins, small envelope (E), matrix (M), nucleocapsid phosphoprotein (N), spike (S), and 16 nonstructural proteins (nsp1-16) that together, ensure replication of the virus in the host cell. Among these proteins, the interactions of N and Nsp3 are essential that links the viral genome for processing. The N proteins reside at CoV RNA synthesis sites known as the replication–transcription complexes (RTCs). The N-terminal of N has RNA-binding domain (N-NTD), capturing the RNA genome while the C-terminal domain (N-CTD) anchors the viral Nsp3, a component of RTCs. Although the structural information has been recently released, the residues involved in contacts between N-CTD with Nsp3 are still unknown. To find the residues involved in interactions between two proteins, three-dimensional structures of both proteins were retrieved and docked using HADDOCK. Residues at N-CTD were detected in interaction with L499, R500, K501, V502, P503, T504, D505, N506, Y507, I508, T509, K529, K530K532, S533 of Nsp3 and N-NTD to synthesize SARS-CoV-2 RNA. The interaction between Nsp3 and CTD of N protein may be a potential drug target. The current study provides information for better understanding the interaction between N protein and Nsp3 that could be a possible target for future inhibitors.Graphic abstract

Cotton leaf curl disease (CLCuD) is a serious constraint to cotton production across Pakistan and north-western India. In the Punjab province of Pakistan at this time CLCuD is associated with one virus, Cotton leaf curl Burewala virus (CLCuBuV), and its associated betasatellite, Cotton leaf curl Multan betasatellite (CLCuMuB). Although some resistance to the virus complex causing CLCuD has been identified in Gossypium hirsutum, this has in the past proven not to be durable. At this time no commercial lines with strong resistance (immunity) to CLCuD are available. Although the cotton fibre produced by Gossypium arboreum is not of the quality required by processors, the species has a number of desirable characters, including resistance to a number of abiotic and biotic stresses, including CLCuD. For this reason there have been efforts to introgress CLCuD-resistance from G. arboreum into G. hirsutum. However, the nature of the resistance of G. arboreum to the viruses causing CLCuD remains unknown. An initial analysis of the resistance was herein conducted by graft inoculation. Graft inoculation of G. arboreum genotype Ravi with scions from severely infected G. hirsutum genotype CIM 496 plants showed that G. arboreum is able to support virus/betasatellite replication and long-distance spread. However, following grafting, only a small number of leaves developing at the time of grafting became mildly symptomatic and symptomatic tissue contained low amounts of virus/satellite DNA, as detected by Southern blot hybridisation; significantly less than the levels seen in G. hirsutum. The symptoms did not spread to the young upper leaves of graft inoculated plants, which contained DNA levels that could only be detected by polymerase chain reaction (PCR) or PCR following an initial amplification by rolling circle amplification–indicating very low DNA levels. The results showed that the resistance of G. arboreum to the virus complex causing CLCuD likely does not involve a mechanism that interferes with delivery of virus by the insect vector. Rather the resistance appears to be due to poor virus replication and possibly interference in short distance (cell-to-cell) spread.

Shoe-string disease caused by Cucumber mosaic virus (CMV) is one of the major threats to tomato production worldwide. The alteration in some biochemical parameters in leaves of the susceptible tomato genotype (Nagina) associated with CMV infection and the effect of exogenous application of salicylic acid (SA) and benzothiadiazole (BTH) were studied in this paper. Results showed that exogenous treatment with SA and BTH not only led to plants which gave significantly more yield than diseased controls (DC), but also delayed symptom expression and reduced disease severity. Analysis of biochemical parameters indicated that exogenous application of elicitors and viral infection, significantly affected the activity of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). Compared to the DC plants, minimum disease severity and maximum number of fruit were recorded after a single dose of SA + BTH. Maximum plant height was recorded after weekly application of SA and maximum fruit yield per plant was gained with single dose of SA. Moreover, the activity of POD was significantly elicited many-fold after weekly application of SA + BTH, while higher amount of SOD was recorded with single dose of SA. The activity of CAT was also significantly accelerated after weekly application of SA + BTH while increased level of APX was noticed with single dose of BTH. In conclusion, the combined application of SA and BTH played an important role in induction of defense mechanism against CMV infection and can be useful in tomato disease management programs.

Four hundred one tomato genotypes representing 11 species were screened against early blight (EB) disease caused by Alternaria solani in a net-house during 2013 to 2017. Considerable variation in resistance/susceptibility levels to EB was observed in the tested germplasm. Only one genotype ‘21396’ f Solanum lycopersicum was resistant and 56 were moderately resistant while the remaining were susceptible to highly susceptible. The pace of disease development was much slower in resistant genotype as symptoms were confined only to lower older leaves. The resistant genotype ‘21396’, most of the moderately resistant ones and a few susceptible and highly susceptible genotypes were rated resistant to stem and fruit infection. Resistant and moderately resistant S. lycopersicum genotypes and mutants with desirable characteristics could be grown in regions with high disease incidence, and can be used for the development of resistant/tolerant cultivars. The moderately resistant germplasm with very poor horticultural traits can nevertheless be used in resistance breeding programmes for developing superior cultivars and hybrids having new resistance genes to EB with a wider genetic base.

Gossypium herbaceum is resistant to cotton leaf curl disease but the nature of the resistance is poorly characterized. The reaction of G. herbaceum cv. Co Tiep Khac to infection by the Burewala strain of cotton leaf curl Kokhran virus (CLCuKoV-Bur) and cotton leaf curl Multan betasatellite (CLCuMuB) was characterized following whitefly-mediated inoculation and grafting. The CLCuKoV-Bur and CLCuMuB isolates used in this study had 97–100% nucleotide sequence identity with other isolates for which sequence information is accessible in databases. A high level of resistance was demonstrated in cv. Co Tiep Khac using 200 whiteflies per plant with no apparent disease symptoms even 90 days post-inoculation, and no detection of the virusbetasatellite complex, as shown by polymerase chain reaction (PCR) and rolling circle amplification (RCA)/PCR. Following graft-inoculation, mild foliar disease symptoms were observed in leaves proximal to the grafting sites of most of cv. Co Tiep Khac plants but not in apical leaves. The presence of CLCuKoV-Bur and CLCuMuB was detected by RCA/PCR but not by PCR and Southern hybridization in symptomless apical leaves of cv. Co Tiep Khac. In addition, less virus accumulated in cv. Co Tiep Khac compared to G. hirsutum cv. CIM-496. These results provided insights into the resistance of G. herbaceum cv. Co Tiep Khac to CLCuKoV-Bur and CLCuMuB.

Black gram [Vigna mungo (L.) Hepper] is an important and nutritionally rich pulse crop mainly grown in tropical and subtropical environments. From July to August 2023–2024, black gram plants in Faisalabad, Pakistan, were observed with severe wilting and root rot disease complex symptoms. Following morphological and molecular characterization, the causal pathogen was identified as Rhizoctonia solani anastomosis group AG-7. Based on the present findings and a review of the literature, this is the first report of R. solani as the causal agent of root rot disease in black gram both in Pakistan and worldwide.

Leaf samples of Solanum lycopersicum exhibiting the tomato leaf curl disease (ToLCD) symptoms were collected from different genotypes. PCR amplifications confirmed the presence of Tomato leaf curl New Delhi virus (ToLCNDV) and betasatellite. Molecular analysis of betasatellite nucleotide sequences showed high level of sequence identity (99%) with chili leaf curl betasatellite (ChLCB) previously reported from chili in Pakistan. To the best of our knowledge, this is the first natural occurrence of ChLCB with ToLCD in Pakistan. Therefore, the association of ToLCNDV with ChLCB is also a component of begomovirus-betasatellite complex that may also affect other cropping systems.